Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Minimal residual disease (MRD) detection and monitoring remains a high challenge for the management of patients with solid tumours 1,2 . A considerable number of patients with breast cancer will develop metastasis within five years of primary tumor resection despite initially being free of detectable metastases depending on the tumor type and stage. In ER(+) breast cancer, after 5 years of adjuvant endocrine therapy, breast cancer recurrences continue to occur steadily throughout the study period from 5 to 20 years 1 . A significant proportion of these early-stage breast cancer patients that seemingly response to treatment have occult micrometastases or MRD that perseveres after initial therapy as a potential source of subsequent metastatic relapse at distant sites. The early identification of MRD in individual patients is highly challenging; towards this goal, real-time highsensitivity liquid biopsy (LB) assays are highly promising and offer a great potential to address this 2 .
LB is a minimally invasive approach that is mainly based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) [3][4][5] . LB is an important tool in the fight against cancer, since it provides the ability to monitor in serial samples a molecular portrait of the tumor in real time 3,6,7 . CTC analysis currently presents a powerful tool for the management of advanced-and early-stage cancer patients; especially CTC molecular characterization offers the unique potential to better understand the biology of metastasis and resistance mechanisms to specific treatments 3,6 . In the early stages of cancer, CTCs are usually detected at very low numbers and are characterized by a high heterogeneity 7 . It is more than ten years that CTCs enumeration performed in the www.nature.com/scientificreports/ in CTCs and two months later progression of disease (PD) was confirmed by detecting metastatic lesions on adrenal gland and liver. During the metastatic phase of the disease, all samples analyzed for CK-19 transcripts were found positive and PD was confirmed a few months later at each time point. This patient died three years after metastasis was documented clinically and by imaging studies and one month after a new progression. Pt#12 was found positive for CK-19 transcripts 61 months since diagnosis and one month later after confirmation of PD (Fig. 1). In Pt#13, no CK-19 transcripts were detected before the initiation of adjuvant chemotherapy. On September 2014, a significant increase in the number of CK-19 transcripts was observed. Eight months later a liver metastasis was confirmed by a liver biopsy. During the metastatic phase of the disease (Fig. 1) all samples analyzed for CK-19 transcripts were found positive.
Detection of ESR1 & PIK3CA mutations. We further analyzed all available plasma-cfDNA and CTCderived gDNA samples from the three breast cancer patients (Pt#11, Pt#12, Pt#13) that developed metastasis at different time points for ESR1 and PIK3CA mutations (Fig. 2). We first used a drop-off ddPCR to detect ESR1 mutations in plasma-ctDNA samples at different time points 41 . Pt#12 was found negative for ESR1 mutations in plasma-ctDNA at all the time points tested; this patient experienced a clinical objective response to endocrine treatment and remained free from disease progression at the time of analysis for 61 months since diagnosis. Pt#13 was found positive for ESR1 mutations in plasma-ctDNA consistently at five different time points during the follow-up period (Fig. 2). This patient later developed resistance to endocrine therapy. In parallel, all available plasma-cfDNA and CTC-derived gDNA samples were analyzed for PIK3CA mutations. PIK3CA E545K mutation was detected in 2/10 gDNA samples from Pt#11 extracted from EpCAM (+) fractions (Fig. 2). Pt#12 was found positive for PIK3CA E545K mutation in 8/14 gDNA samples extracted from EpCAM (+) fractions and in 1/14 paired plasma-ctDNA samples (Fig. 2). Pt#13 was found positive for PIK3CA H1047R mutation in 3/10 gDNA samples extracted from EpCAM (+) cell fractions and in 2/10 paired plasma-ctDNA samples. PIK3CA E545K mutation was detected in 2/10 paired plasma-ctDNA samples.
Comprehensive liquid biopsy analysis for Pt#13. An overview presenting timeline of the disease course from diagnosis onward, therapeutic interventions and the results for CTC enumeration, and the phenotypic and molecular characterization of CTCs at different time points for Pt#13 is shown in Fig. 3A; therapeutic interventions for this patient at different time points are shown in Fig. 3B.

Discussion
We present a comprehensive liquid biopsy analysis of 13 ER(+) breast cancer patients initially diagnosed with operable disease, based on the analysis of serial peripheral blood samples at different time points in a period of ten years, and correlate our findings with the clinical outcome.
In all ten patients that remained free from disease progression during the follow-up period, CK-19 transcripts were detected only at 3/135 (2.2%) time points tested. Until now, all these patients are alive and remained free from progression disease, except Pt#8 that was lost to follow-up. On the contrary, an important increase in the copy number of CK-19 transcripts was observed for two patients (Pt#11, Pt#13) a few years after initial diagnosis and before the confirmation of PD and diagnosis of metastasis. Our results are in agreement with previous clinical studies who have shown that the detection of CK-19 (+) mRNA cells in the peripheral blood of patients with operable breast cancer before, during and after adjuvant treatment is an independent prognostic factor associated with an increased risk of disease relapse and shorter survival [16][17][18]44,45 . It has also been shown that the presence of CK-19 (+) CTCs after the completion of chemotherapy is associated with increased risk of late relapse and poor survival in metastatic breast cancer 46 Our findings indicate that in 8/10 (80%) of early breast cancer patients that did not relapse no CK-19 transcripts were detected in the EpCAM (+) CTC fraction. However, there were two patients P#3 and P#6 that did not relapse during the time of the follow-up. In these two patients we detected CK-19 transcripts in EpCAM (+) CTC fractions, but in only one time point out of 14 (1/14, 7%) time points tested for P#3, and in only two timepoints out of 16 (1/16, 6%) for P#6. It is well known that most of CTCs are destroyed during circulation by immune cells, and this very low detection rate of CTC in peripheral blood could possibly be an explanation for the lack of disease recurrence in these two patients.
Three out of these thirteen patients (Pt#11, Pt#12, Pt#13) developed distant metastasis during the follow-up period. In these patients we further analyzed DNA samples extracted from EpCAM (+) cell fractions and paired plasma for the detection of PIK3CA and ESR1 mutations. ESR1 mutations were not detected in any sample for Pt#11 and Pt#12. ESR1 mutations were detected in the plasma ctDNA of Pt#13 consistently at five different time points during the follow-up period. This patient received everolimus and exemestane for a long time, and after disease progression on this therapy, ESR1 mutations were detected in serial samples of plasma ctDNA. Our results are in accordance with those reported in the EROS1 study, where ESR1 mutations were identified in patients previously treated with tamoxifen or aromatase inhibitors, revealing a possible correlation between long term aromatase inhibitor therapy and the existence of ESR1 mutations 49 . Additional studies have shown that acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors [50][51][52][53]  www.nature.com/scientificreports/ indicating that ESR1 mutations (especially D538G, Y537S) are associated with more aggressive disease 38,53,54 .
In the phase III PADA-1 trial presented at 2021 San Antonio Breast Cancer Symposium, it was observed that switching from an aromatase inhibitor plus palbociclib to fulvestrant and palbociclib upon early identification of the ESR1 mutation in plasma-before disease progression-the median PFS was doubled. This trial has also shown that ESR1 mutations are rarely detected in plasma-cfDNA of ER+ HER2− MBC patients with no overt resistance to aromatase inhibitor and that the detection of ESR1 mutations was associated with a significantly shorter PFS, suggesting that the existence of ESR1 mutation at baseline could accelerate the outset of resistance to AI-palbociclib 55 . The first results of this trial have recently been published showing that the early therapeutic targeting of ESR1 mutations in blood results in significant clinical benefit 56 . It is an important study as the original design explored in PADA-1 might help with tackling acquired resistance with new drugs in future trials 56 . In our study, PIK3CA mutations were detected in all patients that later developed metastasis. More specifically in Pt#11, E545K mutation was detected in 1/10 (10%) EpCAM (+) CTC fractions but not in paired plasma-ctDNA samples. In Pt#12, E545K mutation was detected in 8/14 (57%) EpCAM (+) CTC fractions and in 1/14 (7%) paired plasma-ctDNA samples checked. In Pt#13, E545K mutation was detected in 2/10 plasma-ctDNA samples but not in paired EpCAM (+) CTC fractions. In the same patient, H1047R mutation was detected in 3/10 (30%) EpCAM (+) CTC fractions and in 2/10 (20%) paired plasma-ctDNA samples. We have already shown before in a direct comparison study using the same blood draw, and the same detection methodology that there is no a 100% concordance in the detection of PIK3CA mutations in CTCs and corresponding ctDNA 31 . In addition, this discordance may be explained as a result of potential intratumor heterogeneity and as an effect of therapeutic pressure on different cancerous subclones.
We further focused our analysis on Pt#13 that was diagnosed with operable BC in 2010 and died from metastatic breast cancer in 2020. CTC enumeration revealed a bad prognosis for this patient, even before the initiation of systemic chemotherapy, that was based on the relatively high number of CTCs. During the first five years (2010-2015) of follow-up, even before metastasis in the liver was histologically confirmed in September 2015 (time point: 69 months), the patient was constantly positive for the presence of CTCs, CTC counts were constantly more than 5CTCs /23 mL PB and a dramatic rise in CTC counts in September 2014 (timepoint: 57 months) has clearly suggested disease progression at least four years before imaging documentation of metastasis.
Rapid increases in CTC numbers at months 74 and 122, were associated with metastatic disease documented by biopsy 6 months earlier. In this patient, verification of recurrence and administration of systemic therapy was verified by tissue biopsy of the metastatic lesions in the liver and was not based on CTC detection. The increase of the CTC number was associated with clinically and radiologically documented disease progression and the different administered treatments were given according to the national guidelines using approved drugs and combinations.
Molecular characterization of CTCs isolated from this patient by double immunofluorescence demonstrated that practically all the detected cells were CK(+)/HER2(+) and CK(+)/Vimentin(+), while the primary tumor was HER2 negative. Based on our initial data demonstrating that trastuzumab can eliminate HER2 + CTCs 57 , the patient was enrolled in an open-label randomized Phase II trial of secondary adjuvant trastuzumab in early-stage breast cancer patients harboring HER2 + CTCs after the completion of adjuvant chemotherapy and radiotherapy 42 and trastuzumab administration resulted in a clear decrease of the CTCs numbers lasting for 8 months. However, at the time of relapse the significant increase of CTCs was characterized by the presence of exclusively HER2-but EGFR+ CTCs. It is to note that lapatinib also resulted in decreased number of CTCs but for a short period of time. Retrospective molecular analysis of DNA derived from CellSearch cartridges, (P#13, time point: 59 months, Nov 2014) has revealed the presence of the E545K PIK3CA mutation in CTCs that is known to confer resistance to trastuzumab as well as ESR1 mutations (D538G, Y537C), that are now known to confer resistance to the everolimus/exemestane combination. In September 2015 (time point: 69 months), CTC number increased again while imaging studies revealed multiple liver metastatic lesions which were histologically confirmed. CTC enumeration has indicated metastasis at least one year earlier (September 2014, time point: 57 months) than radiologically revealed and histologically confirmed (September 2015, time point: 69 months).
It is remarkable that even one year before clinical manifestation of metastasis, and till 2020, PIK3CA hotspot mutations (E545K and H1047R) and ESR1 mutations (D538G, Y537C, Y537S, Y537N), were continuously detected, not only in CTC-derived DNAs from CellSearch cartridges, but also in plasma-cfDNA, and EpCAM (+) CTC fractions as well. At the same time period (Feb 2016-May 2020), ESR1 methylation, that was also shown to be associated with lack of response to hormonotherapy 41 , was detected in plasma-cfDNA and corresponding EpCAM (+) CTC fractions, but not in CTC-derived DNAs from CellSearch cartridges. Based on these results, it is evident that while the rise in CTC counts indicated very early the metastatic spread, CTC molecular characterization at the DNA level clearly indicated that the tumor had already developed resistance to the treatment. During Feb 2016-May 2020 gene expression analysis in EpCAM (+) CTCs fractions, has revealed the EMT nature of these cells, since the epithelial marker CK-19 that was expressed in all samples tested, was co-expressed with stem cell markers and EMT markers. CD24 -/low /CD44 high profile was detected in all samples and the CD24 −/low /ALDH1 high in 50% of samples analyzed during this period of time. Our results on CK-19, TWIST1, CD24 and ALDH1 expression in CTCs are in accordance to those presented in a previous study 58 .
Molecular characterization of CTC at the DNA level revealed one year before the clinical manifestation of metastasis the presence of PIK3CA and ESR1 mutations in CTCs that are now known to be highly important for therapy resistance. Our retrospective analysis indicates that these findings were an early indication that disease would not respond to the therapy given to this patient (Trastuzumab, lapatinib, and Afinitor/Aromacin), but at that time this was not known, and moreover Alpelisib was not FDA-approved. Alpelisib was administered to P#13 for 2 months, but severe adverse events (diabetes and septicemia necessitating patient's hospitalization for 18 days) resulted to early treatment discontinuation. The patient died from disease progression 6 months later. www.nature.com/scientificreports/ To the best of our knowledge, this is the first time that early detection of Minimal Residual Disease in breast cancer based on a comprehensive liquid biopsy analysis and a ten year follow-up of operable breast cancer patients is reported. According to our results, CTC analysis provided highly important information for the management of these patients. CTC enumeration as expected indicated at the very beginning whether these patients were at a very high risk for metastatic spread. Our results indicate that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients, and could guide oncologists to select molecular targeted treatments, based on precision medicine. Up to now, the use of CTCs to guide systemic therapy remains controversial, and there are no definitive data to support its clinical utility. However, a number of clinical trials based on liquid biopsy are ongoing and are expected to demonstrate the clinical utility of CTCs 59 . Our results indicate that comprehensive liquid biopsy analysis could reveal the presence of MRD four years before the appearance of clinically detectable metastatic disease. We strongly believe that interventional clinical trials based on a comprehensive liquid biopsy analysis are highly needed to change clinical practice for the benefit of breast cancer patients.

Methods
Patients. All patients were diagnosed with early and operable ER(+) breast cancer. Peripheral blood (PB) (20 mL) was obtained from all patients at different time points during a long-term follow-up period since diagnosis, two weeks after the removal of the primary tumor. Ten of these patients did not relapse and remained metastasis free during the follow-up period, while three of them relapsed within 5-7 years. In all PB samples EpCAM (+) cell fractions were enriched using immunomagnetic capture beads, and analyzed for CK-19 mRNA expression, and in parallel, ctDNA, isolated from paired plasma using the same blood draw (2 mL), was analyzed for all these patients. All samples were analyzed for ESR1 and PIK3CA mutations in genomic DNA (gDNA), extracted from EpCAM (+) fractions and in paired plasma ctDNA at different time points. CTC enumeration using the CellSearch system (Menarini, Silicon Biosystems) was also performed. For one patient that later developed metastasis, CellSearch analysis was performed at regular intervals within a year for 10 consecutive years and phenotypic characterization of CTCs was also performed through double immunofluorescence. gDNA was extracted from the isolated CTCs from CellSearch cartridges to detect ESR1 and PIK3CA mutations for this patient and all CTC-derived gDNA samples were also analyzed for ESR1 methylation status. All patients have signed informed consent for MRD screening, and the study was conducted in accordance with the Declaration of Helsinki and has been approved the Medical Ethical Committee of the General University Hospital of Heraklion, Crete, Greece (Ethical Allowance: 8756/23-6-2014).
Molecular analysis of CTCs. Gene expression. RNA isolation: cDNA synthesis. All steps including, the isolation of EpCAM (+) fractions, total RNA extraction using Trizol-LS reagent (Invitrogen, USA) were performed as previously described 15,60,61 . cDNA synthesis was carried out with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA) according to the manufacturer's protocol in 20μL of total volume reaction. All cDNA samples were kept at − 20 °C until use.
RT-qPCR. RT-qPCR was performed for the following genes: (a) CK-19, (b) EMT-associated marker TWIST-1, and (c) stem-cell markers CD24, CD44, ALDH-1. B2M (beta-2-microglobulin) was used as a reference gene 61 . RT-qPCR assays for the quantification of CK-19, TWIST-1, CD24, CD44 and ALDH-1 transcripts were performed as previously described 14,15,62 . The cut-off for CD24, CD44 and ALDH-1 was estimated in respect to HPRT expression as previously reported 61 in a group of 10 healthy individuals whose peripheral blood has been analyzed in exactly the same way as patient's. DNA analysis. DNA isolation. Genomic DNA (gDNA) was isolated from CellSearch cartridges, and EpCAM (+) CTC fractions at regular timer intervals from initial diagnosis during the follow-up period as follows: (a) CellSearch cartridges: The pre-enriched sample (pre-stained CTCs and WBCs) was aspirated from the corresponding CellSearch cartridges, gDNA was extracted using the QIAamp DNA Micro Kit (Qiagen, Germany) in accordance with manufacturer's instructions 42 . (b) EpCAM (+) fractions: Similarly, gDNA from all available EpCAM (+) fractions was extracted from CTCs in Trizol LS (Invitrogen, USA) as previously described 30,63 . In parallel to the isolation of CTC-derived gDNA, plasma cell-free DNA was isolated as previously described 31 . The QIAamp Circulating Nucleic Acid Kit (Qiagen) was used to isolate ctDNA from 2.0 mL of plasma according to the manufacturer's instructions. DNA concentration was measured in all samples, using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA) and DNA integrity was assessed prior to the analysis by amplifying a wild-type region in exon 20 of PIK3CA gene 64 .
ESR1 methylation. All available DNA samples were also processed to sodium bisulfite (SB) treatment using the EZ DNA Methylation Gold Kit (ZYMO Research Corp., USA) according to manufacturer's instructions. Only samples that were positive for exon 20 PIK3CA amplification were further processed to SB-treatment. SB-treated www.nature.com/scientificreports/ DNA was stored at − 80 °C until further use. After the SB-treatment, SB-converted DNA integrity was assessed by a real-time methylation-specific PCR (MSP) for b-actin (ACTB) as previously described 41,60 . Subsequently, all SB-treated samples were analyzed for ESR1 promoter methylation using real-Time MSP, based on our previously developed and validated protocols 41,66 . CTC enumeration. CTC enumeration was performed using the FDA-cleared CellSearch system (Menarini, Silicon Biosystems) in PB samples collected during all these years at regular time intervals for most patients. For Pt#13 CellSearch analysis was performed for 10 consecutive years in peripheral blood draws in sequential patient samples During the first 5 years of follow-up, before the development of clinically confirmed metastatic disease, 23 ml of peripheral blood collected in CellSave preservative tubes was analyzed. Conversely, during the metastatic phase of Pt#13 disease 7.5 mL of peripheral blood were used for CTC enumeration every 3-6 months.
Double immunofluorescence (IF). Phenotypic characterization of CTCs was performed only for Pt#13.
CTCs isolated from 20 ml of peripheral blood were used for their phenotypic characterization during the followup. The first 5 mL of blood were discarded to avoid contamination with epithelial cells from the skin. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient (d = 1.077 g/mL) centrifugation at 1800 rpm (600 g) for 30 min. Cytospins of 5 105 PBMCs were prepared and stored at − 80 °C until their use for double staining experiments. The presence of CK-positive cells in PBMCs' cytospins was investigated using the A45-B/B3 mouse antibody (anti-CK-8, CK-18, CK-19, Micromet Munich) and is further referred as CK+ in the text. At some time points the presence of CTCs in PBMCs' cytospins was investigated using monoclonal antibodies against ki67 (proliferation marker, Abcam) or/and M30-FITC conjugated (apoptotic marker, Roche Diagnostics, Basel). Double immunofluorescence staining was performed as previously described [67][68][69][70][71][72] .

Data availability
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical restrictions.